Accurate detection and characterization of lymph nodes that receive drainage from a primary cancer [i.e., sentinel lymph nodes (SLNs) and secondary nodes (2Ns)] has a direct impact on surgical management and the choice of treatment options. Two methods are currently used to identify SLNs;peritumoral injection of radioisotopes followed by either scintigraphy or gamma probe evaluation and injection of dye with detection of dye-stained SLNs at surgery. Currently, there are no reliable means to identify 2Ns. When a SLN contains metastases the common practice is to perform radical lymph node surgery followed by pathology of resected nodes to evaluate them for the presence of metastases. Investigations using a melanoma swine model have demonstrated the superior accuracy of contrast-enhanced ultrasound (CEUS) compared to scintigraphy for the detection of SLNs. Research has also confirmed that CEUS can be used as a reliable method to identify metastases in SLNs. Recent preliminary research suggests that CEUS can also be used to identify 2Ns including those that contain metastases. The current proposal will expand previous research to evaluate the use of CEUS as a means to detect 2Ns in the melanoma swine model. The hypothesis is that CEUS will permit detection of the SLN's efferent lymphatic channels, which will allow detection of contrast-enhanced 2Ns. The specific aims are: 1) To determine if CEUS can be used to identify 2Ns in swine with melanomas;and 2) To compare the rate of tumor detection in 2Ns identified by CEUS to the rate of tumor detection in 2Ns obtained by conventional radical lymph node resection. A secondary aim is to correlate the amount of metastases in SLNs to the number of 2Ns that contain metastases and the amount of metastases in 2Ns. thirty melanoma-bearing swine will be studied. A reticuloendothelial system-specific ultrasound contrast agent (UCA) will be injected around each melanoma followed by CEUS to detect the contrast-enhanced SLNs. Then, the UCA will be injected directly into the SLNs and CEUS will be used to identify contrast-enhanced lymphatic channels leading from the SLN to enhanced 2Ns. Blue dye will be injected around the melanoma tumor and a surgeon (blinded to the results of CEUS) will use the blue dye to guide resection of the SLNs. The surgeon will also perform radical surgery of any nodes that they believe represent 2Ns. All resected nodes will be submitted to pathology to determine if they contain metastases. The rate of tumor detection in the 2Ns identified by CEUS will be correlated to the rate of tumor detection in nodes removed by radical resection using pathology as the gold standard. The amount of metastases in the SLNs will be compared to the number of 2Ns that have metastases and the degree of tumor involvement in the 2Ns. The potential benefits of this innovative study will be the development of a minimally-invasive CEUS imaging method to map lymphatic drainage from melanomas which will allow precise localization of both SLNs and 2Ns thereby obviating, or significantly reducing the need to perform radical lymph node resections and potentially reducing surgical complications. PUBLIC HEALTH RELEVANCE: establishing the spread of tumor into regional lymph nodes is critical for the surgical management of patients with cancer. Hence, this project will develop a novel, minimally- invasive, safe, diagnostic contrast-enhanced ultrasound imaging method to detect lymphatic drainage from melanoma tumors, which will allow regional lymph nodes to be identified. Such an imaging technique should significantly increase the ability to detect the spread of cancer and reduce surgical side effects for an overall improvement in cancer treatment.